WO2005001091A1

WO2005001091A1 - Probe set for detecting mutation and polymorphism in nucleic acid, dna array having the same immobilized thereon and method of detecting mutation and polymorphism in nucleic acid using the same

Info

Publication number: WO2005001091A1
Application number: PCT/JP2004/008496
Authority: WO
Inventors: Hiroko Sakamoto
Original assignee: Olympus Corporation
Priority date: 2003-06-27
Filing date: 2004-06-10
Publication date: 2005-01-06
Also published as: JPWO2005001091A1

Abstract

It is intended to provide a probe set whereby a mutation in various sequences can be detected by a difference at the single nucleotide level with showing a significant difference in a DNA array under the same conditions. Namely, a probe set wherein a site corresponding to the mutation to be detected is located at the center of a probe, sequences completely identical with the target nucleic acid to be detected are provided on both ends thereof and universal base(s) are optionally provided in the completely identical sequences.

Description

Description Nucleic acid mutation and polymorphism detection probe set,

A DNA array on which it is immobilized,

And methods for detecting mutations and polymorphisms using them

The present invention relates to a probe set for detecting a mutation or polymorphism of a nucleic acid, a DNA array having the same immobilized thereon, and a method for detecting a mutation or polymorphism using the same. Background art

It is known that mutations in the nucleotide sequence of a specific gene on the genome change the structure and expression level of the protein encoded by the gene and cause various diseases such as cancer. More recently, single nucleotide polymorphisms (Single

It has become clear that Nucleotide Polymorphism (SNPs) causes differences in drug susceptibility and in the prevalence of certain diseases in humans. It has also become clear that bacteria are correlated with drug resistance. Therefore, detecting mutations in the genomic nucleotide sequence can be used not only to diagnose diseases, but also to measure disease morbidity, predict individual susceptibility to drugs and side effects, and predict bacterial drug resistance. It is suggested that providing appropriate medical treatment to individuals, that is, the so-called order-made medical treatment is essential.

For example, it has become clear that such SNP sites exist on the human genome at a frequency of one in hundreds of bases throughout the genome. In order for the information of these SNPs to be applied to actual medical treatment, a technology that enables accurate, simple, and low-cost detection of as many SNP sites as possible for each individual is required.

Conventionally, a nucleotide sequence decoding method based on the Didoxy method and the Sanga method has been used as the most reliable method for detecting mutations in the nucleotide sequence. These methods make it possible to determine not only the presence or absence of mutation but also the typing of mutation in the case of base substitution. However, it is a technique that can determine even deletion or insertion of a base with high accuracy. However, the number of mutation sites that can be detected in one reaction is limited to a range of several hundred bases consecutively on the genome.In particular, multiple SNPs scattered several hundred bases or more apart in the genome Is difficult to detect at the same time. Therefore, in the case of the above-described simultaneous analysis of mutations which requires simultaneous processing, there are difficulties in terms of simplicity and cost.

In recent years, a technique called a DNA array (or a DNA chip) has attracted attention as a means for detecting base sequences scattered in such a wide range of genomes simultaneously and in parallel. The DNA array is obtained by immobilizing various types of oligonucleotide probes on a carrier such as a silicon wafer or a slide glass to separate fractions, respectively. Under a certain condition, the labeled sample nucleic acid is brought into contact with the DNA array to carry out an hybridization reaction with the probe, and the presence or absence and degree of the hybridization reaction in each fraction is determined from each fraction. It is measured by detecting and comparing signals, and is used to detect the presence or absence of a specific base sequence in the nucleic acid sample and its expression level.

In principle, DNA arrays can detect multiple SNPs located several hundred bases or more apart in the genome at the same time in parallel and in large quantities, so individual SNP detection, that is, mutation detection It can be used for analysis. Therefore, it is thought that the use of a DNA array for mutation analysis will contribute to the realization of the above-mentioned order-made medical treatment. However, there are still issues to be solved.

The first challenge in applying DNA arrays to the simultaneous analysis of multiple mutations is to develop a probe sequence design method to detect various sequences under the same conditions with a significant difference at the level of one base. It is to be. Probes for simultaneously detecting various nucleotide sequence variations in the genome in a DNA array require various properties. For example, when multiple types of probes prepared to detect various different sequences are hybridized with the target nucleic acid corresponding to each probe, the optimal probe for each probe is used. The hybridization conditions are considered to have a certain extent. And there is usually a significant difference between the Tm's of the probes. When performing gene expression analysis on a DNA array, the probe If the region in the target sequence to be hybridized is selected in consideration of the Tm value of the probe within the target sequence, the Tm value can be adjusted to a relatively narrow range among various probes used on the same array. It is possible. However, in the case of mutation analysis, unlike in the case of expression level analysis, the necessity of preparing a probe that hybridizes to the region containing the mutation site to be analyzed requires flexibility in changing the Tm value. Is considered to be relatively low. Therefore, if measurements using probes with different Tm values are performed under the same conditions, a group of samples that will be measured outside the optimal condition range will be included in the DNA array. Therefore, data including false positive and false negative results may be obtained, which is not preferable.

Furthermore, even if the same detection region includes a plurality of mutation sites, for example, two mutation sites, the relationship between the probe and the detection region may be roughly classified into a perfect match and one base. There are at least three types of mismatches and two-base mismatches. In the case of a mismatch, it can be considered that there are a plurality of types (combinations) of mutations, if they are further classified. Therefore, it is important to distinguish at least a perfect match, a single-base mismatch, and a two-base mismatch, and if possible, it is desirable that the type of mutation can be clearly distinguished. Probes used to detect SNPs in DNA arrays must meet these requirements. However, at the same time as distinguishing a perfect match from a single base mismatch, it is also difficult to clearly distinguish between a single base mismatch and a two base mismatch depending on the measurement temperature. In particular, when a plurality of mutation sites are close to each other, it is difficult to distinguish a perfect match, a single base mismatch, and a two base mismatch. The appropriate temperature to detect the difference between a perfect match and a single-base mismatch hybridization is often unsuitable for detecting the difference between a single-base mismatch and a two-base mismatch hybridization. is there. The reverse is also true. Therefore, keeping the Tm value of a probe set prepared for detecting a set of mutations in the same region within a narrow range in all the probe sets on a DNA array is particularly important when multiple mutations are close to each other. In the past, it was very difficult or impossible to do so. In order to realize customized medicine using DNA arrays, it is necessary to address these issues. One guideline for designing a probe sequence for mutation detection is described in, for example, the following literature.

Chee M. et al., "Science", (USA), 1996, 274, pp. 61-10-14.

The above documents provide general guidelines for a probe sequence designing method for mutation detection, but as described above, in particular, probe design guidelines for simultaneously and accurately detecting a plurality of adjacent mutations. Does not give. Disclosure of the invention

Means for Solving the Problems The present invention is configured as described below in order to solve the above problems and achieve the object.

The probe set of the present invention is a probe set for detecting a mutation or polymorphism in a base sequence, and each probe in the probe set has an odd number of all bases N of the probe ( N = 2n + 1, where n is a natural number; the same applies to the following), the base a (complementary to the mutation X present in the target sequence) is present at the base (n + 1th from both ends) of the probe If the total number of bases N of the probe is even (N = 2n + 2), one of the two bases at the center (n + 1st from the 5 'end and 3' end, respectively) One of the bases is provided with a base a complementary to the mutation X present in the target sequence; and the 5, terminal sequence and the 3 ′ terminal sequence of the base a complementary to the mutation X Contains a sequence that is completely complementary to the target sequence

It is characterized by that.

In the probe set of the present invention, when there is a possibility that at least one mutation Y other than the mutation X is contained in the target sequence, each probe for the target sequence depends on the type of the base of the mutation Y. It is preferable to have a universal base Z that forms a base pair with the base b of the mutation Y at a site corresponding to the mutation Y without performing the above.

In the probe set of the present invention, when a mutation X and a mutation Y ( _{p = 1} to _q ) (Q is an integer of 1 or more corresponding to the number of mutations other than the mutation X) are present in the target sequence. The probe set to use,

(1) a probe for detecting the mutation X or a polymorphism caused by the mutation X, which has a base a complementary to the mutation X at a site corresponding to the mutation X, and wherein the mutation Y ( _{p = 1} to _q ) at a site corresponding to at least one mutation, a universal base that forms a base pair with the base in the at least one mutation without depending on the type of base of the at least one mutation A probe for detecting mutation X having Z; and

(2) at least one mutation in the mutation Y _{(p = 1} ^ _q) or a mutation or polymorphism detection probe for detecting a polymorphism due to the at least one mutation, or detection of the mutation or polymorphism Probe set for

At a site corresponding to at least one mutation in the mutation Y ( _{p = 1} to _q ), there is a base complementary to the base of the mutation, and the mutation X and the mutation Y ( _{p =}卜). A probe or probe set having a universal base Z in at least one site corresponding to a mutation other than at least one mutation therein;

It is characterized by including.

In the probe set of the present invention, the universal base Z is desirably selected from the group consisting of inosine, 5-nitroindole, and 3-nitropyrrol.

The probe set of the present invention includes, when there is a possibility that at least one mutation Y other than the mutation X may be contained in the target sequence, each probe for the target sequence is provided at a site corresponding to the mutation Y. It is also possible to form an aggregate of probe molecules containing any of the nucleotides of adenine, guanine, cytosine, and thymine at random (substantially the same amount).

The probe set of the present invention is characterized in that, when the target sequence contains a mutation X and a mutation Y ( _{p = l} to _q ) (q is an integer of 1 or more corresponding to the number of mutations other than the mutation X), A probe in which any of the nucleotides of adenine, guanine, cytosine, and thymine is randomly included at a site corresponding to at least one of the mutations Y ( _{p = l} to _q ) The feature is that it is an aggregate of molecules. In the probe set of the present invention, between the mutation X and the mutation Y or at least one of the mutations Y ( _{p = 1} to _q ), 0 to 10 bases Is preferably present.

The probe set of the present invention has at least one probe in the above probe set in which a base complementary to the mutation X is at the center of the probe and other bases are completely complementary to the sequence of the target nucleic acid. (Detect probe) and at least one probe (reference probe) having a sequence identical to that of the detect probe in a region where a mismatch occurs at the site corresponding to the mutation X described above and in the other region. It is characterized by the following.

In addition, the probe set of the present invention includes at least one probe (detector) in the above probe set which is used particularly when there is a possibility that at least one mutation Y other than the mutation X is contained in the target sequence. A) a mismatch occurs at a site corresponding to the mutation X, and at least a mutation other than at least one mutation in the mutation Y or the mutation 変異 ( _{ρ =} to _q ) One of the sites includes the universal base Z, and the other region comprises at least one probe (reference probe) having the same sequence as the detect probe.

In these probe sets, it is preferable to include a universal base Z at the site corresponding to the mutation Y. In particular, the universal base Z is preferably inosine, 5-nitroindole, and 3-nitropyrrole. Can be selected from the group consisting of

In these probe sets, adenine, guanine, cytosine, and a mutation Y, or at a site corresponding to at least one mutation other than at least one mutation in the above-described mutation Y ( _{P = 1} to _Q ), It is also possible to form an aggregate of probe molecules containing any of thymine nucleotides at random.

In the probe set of the present invention, n defining the total number of bases (N = 2n + 1 or N = 2n + 2) of each probe in the probe set is in the range of 5 to 18. As a result, it is desirable that the Tm of each probe falls within a desired value V-10. The DNA array of the present invention is one in which any of the above probe sets is immobilized.

The method for detecting a mutation or polymorphism in a target nucleic acid according to the present invention comprises the steps of: detecting a target on a solid-phased DNA array when any of the above probe sets does not include a set of a detector probe and a reference probe; The nucleic acid is brought into contact under optimal conditions with the hybridization between each probe in the probe set and the target nucleic acid, the signal from each probe is quantified, and then, optionally, Either increase or decrease the temperature by +/- 10 ° C from the average Tm of the probe, and then further quantify the signal, or use any of the above probe sets Contains a set of a detector probe and a reference probe, the target nucleic acid is transferred between each probe in the probe set and the target nucleic acid on the solid-phased DNA array. Detection of mutations or polymorphisms in the target nucleic acid, by contacting the hybridization under optimal conditions and quantifying and comparing the signals from the detector probe and the reference probe. Is the way. Brief Description of Drawings

FIG. 1 is a conceptual diagram showing the thermal stability of a target nucleic acid-probe complex in a state of perfect match, single base mismatch, and two base mismatch at various temperatures under the same salt concentration condition.

FIG. 2 is a diagram showing the results of one example of the present invention.

FIG. 3 is a diagram showing an example of a probe set necessary for detecting two adjacent mutations using a conventional probe set.

FIG. 4 is a diagram showing an example of a detection result when two adjacent mutant portions are actually detected using the conventional probe set of the type shown in FIG.

FIG. 5 is a diagram showing an example of a probe set when two adjacent mutation sites are detected by the probe set of the present invention using a universal base.

FIG. 6 is a diagram showing an example of a detection result in a case where two adjacent mutant portions are actually detected using the probe set of the present invention shown in FIG. BEST MODE FOR CARRYING OUT THE INVENTION

As used herein, the term “nucleic acid” refers to any of DNA, DNA including artificial nucleotides, RNA, and RNA including artificial nucleotides, PNA, and PNA including artificial nucleotides.

The probe set of the present invention is a probe set for detecting a mutation or polymorphism in a nucleotide sequence, and each probe in the probe set has an odd number of all bases N of the probe. (N = 2 n + l, where n is a natural number; the same applies to the following), the base at the center of the probe (n + 1 from both ends) is complementary to the mutation X present in the target sequence a If the total number of bases N of the probe is even (N = 2n + 2), any of the two bases at the center (n + 1st from the 5 'end and 3' end, respectively) At either one of the bases, a base a complementary to the mutation X present in the target sequence is arranged; and the 5′-end sequence and the 3′-end sequence of the base a complementary to the mutation X are both It is characterized by including a sequence that is completely complementary to the target sequence.

It is desirable that there is a perfect match between each probe used here and the sequence it recognizes.However, in some cases, the target nucleic acid contains no mutation, resulting in a single base mismatch. They can be in a relationship. This is the case when the mutation X, which was assumed to be complementary to the base a in the probe, is actually a wild type, and as a result, a mismatch occurs in that portion, or This is because there is a possibility that the mutations are different. In such a case, a probe that perfectly matches the wild-type sequence or a probe that perfectly matches a base that is neither wild-type nor mutant X should be included in the probe set in advance. A positive reaction will result in any of these probes. As a result, it is determined whether or not the site includes the mutation X, a mutation other than the mutation X, or a wild type.

In order to distinguish between the mutant type and the wild type, a probe set including a detection probe and a reference probe according to one embodiment of the present invention may be used. The probe set of this embodiment includes at least one of the above probe sets. 1 probe (detector probe) and at least one probe (reference probe) having a mismatch at a site corresponding to the mutation X and having the same sequence as the detectable probe in the other region. Consisting of The detect probe contains a base a complementary to the mutation X. For example, if this is a guanine (G) base, the corresponding base of the reference probe is at least one of (:, T, Α). And preferably a probe set in which all of these are set in triplicate.

As described above, each probe in the probe set of the present embodiment is preferably used in a relationship between the target nucleic acid and the probe where a perfect match or a single base mismatch is assumed, but may include a separate mutation in some cases. It is possible to use it in the state where it is out of the way. In such an embodiment, at least three relationships of a perfect match, a single base mismatch, and a two base mismatch are possible. These states and their detection will be described below with reference to the drawings.

In this figure, the S-shaped solid line shows the temperature change of the complex (T m == T m (PM)) in which the entire region of the probe perfectly matches the target nucleic acid. It shows how the double-strand formation rate of the probe-target nucleic acid complex changes when the reaction is performed. On the other hand, the S-shaped dotted line immediately below the solid line indicates that a single-base mismatch is included (T m = TM (MM 1)), and the S-shaped dotted line further below indicates a two-base mismatch. Include (T m = T m (MM 2)). In general, the thermostability of probe-target nucleic acid complexes is higher for perfect matches than for incomplete matches (single- and double-base mismatches), but there is a large difference in temperature .

In Fig. 1, when the temperature is in the low temperature region of a, the case of perfect match and the case of incomplete In the case of all matches, the double strand formation rate is high and it is difficult to discriminate. In this temperature region, non-specific binding tends to occur, so there is no big difference between perfect match and incomplete match. On the other hand, when the temperature is in the high temperature range of c, contrary to the case of the temperature a, the probe and the target nucleic acid tend to separate from each other in both perfect match and incomplete match. It is difficult to identify each other. Furthermore, the low level of the signal also contributes to this temperature range, making it unsuitable for measurement.

On the other hand, when the temperature is in the region of b, it can be seen that the perfect match and the incomplete match can be easily distinguished by the difference in the duplex formation rate. When looking at this figure in the vertical axis direction, the distance between the solid line and the dotted line is not only longer than in the temperature regions a and c, but also the perfect match and the single base mismatch. It can be seen that a significant difference may occur between the difference between the 1-base mismatch and the 2-base mismatch. For example, since d and e in FIG. 1 are significantly different in length, the duplex formation rate is significantly different, which can be identified as a significant difference in signal intensity from the DNA array. This means that a perfect match can be distinguished from a single base mismatch. However, this means that at this temperature _Tfl, it is difficult to discriminate between a one-base mismatch and a two-base mismatch (substantially difficult or large uncertainty).

The fact that a perfect match, a single-base mismatch, or a two-base mismatch actually occurs is described below with reference to specific sequences.

The nucleotide sequence shown in SEQ ID NO: 1 in Table 1 shows the wild-type sequence of the target nucleic acid to be detected, and SEQ ID NO: 2 and SEQ ID NO: 3 show the single nucleotide variant including mutation X and mutation Y, respectively. Is shown. Mutation X is a mutation in which the base in the wild type was changed to G, while mutation Y is a mutation in which the base in the wild type was changed to A in T. Target nucleic acid and sequence of o-a

The sequences described in SEQ ID NOs: 4 to 7 are a set of probes for detecting mutation X. The sequence described in SEQ ID NO: 4 is a probe that perfectly matches the mutation X, and SEQ ID NOs: 5 to 7 are single nucleotide mismatches with the target nucleic acid. Among them, the sequence shown in SEQ ID NO: 5 does not contain the mutation X, and matches perfectly when it is a wild type.

The sequences described in SEQ ID NOS: 8 to 11 are a set of probes for detecting the mutation Y. The sequence described in SEQ ID NO: 8 is a probe that perfectly matches the mutation Y. SEQ ID NOS: 9 to 11 are single-base mismatches with the target nucleic acid. Among them, the sequence shown in SEQ ID NO: 9 contains mutation Y. It is a perfect match if it is wild type.

When these sets of probes are immobilized on a DNA array and the mutation analysis of the two sample nucleic acids (target nucleic acids) shown in Table 2 is performed under the same conditions, each probe and the mutation to be detected The relationship between the numbers is as shown in Table 3.

Table 2 Sequence of target nucleic acid in sample

Table 3 Number of mis-merits ^per product between sample and product

As shown in Table 3, the relationship between the target nucleic acid and these probe sets is either a perfect match or an incomplete match, and in the case of an incomplete match, one more base There may be a mismatch and a two-base mismatch. In the case of sample A, it is a mutant containing mutation X but not containing mutation Y, that is, a wild type. On the other hand, sample B is a mutant containing mutation X, but is a sample containing mutation Y in one sequence.

In sample A, the relationship between the target nucleic acid and the probe is limited to a perfect match and a single-base mismatch. If so, mutation analysis is achieved. On the other hand, in sample B, the relationship between the target nucleic acid and the probe is not a perfect match, but may be a one-base mismatch or a two-base mismatch. As described above, the measurement conditions suitable for discriminating between a perfect match and a single-base mismatch are not necessarily the measurement conditions suitable for discriminating between a single-base mismatch and a two-base mismatch. It can be understood that it is difficult to perform the procedure accurately on the DNA array under the same conditions. In the case of a conventional probe set, as described above, it has been difficult to simultaneously and clearly identify a perfect match, a single base mismatch, and a two base mismatch. However, it can be seen from FIG. 1 that the use of the probe set of the present invention makes it possible to discriminate between a one-base mismatch and a two-base mismatch by shifting the measurement temperature to a lower temperature. Because the length of g is longer than the length of e, and the length of f is also large enough.) Therefore, even if two mutations are contained in the target nucleic acid as a result, if the measurement temperature is slightly changed as much as possible using a probe set consisting of a detector probe and a reference probe, a perfect match and one base It is possible to distinguish not only between mismatches, but also between 1-base mismatches and 2-base mismatches.

To do so, n, which defines the total number of bases in the probe (N = 2n + 1, or N = 2n + 2), is in the range of 5 to 18, and as a result, each probe in the probe set It is desirable that Tm is included in a range of a desired value + 10 ° C. If the Tm value is specified in this way, the Tm value of all the probes in the probe set falls within a much narrower range than the conventional probe set. Can cope with the above-mentioned two-base mismatch by slightly changing the measurement temperature, making it possible to specifically detect mutations under essentially the same conditions. .

Therefore, the probe set of the present invention does not contain the expected mutation, and further contains the unexpected mutation at another site, that is, it contains two convenient mutations. Is also potentially available. However, it is already known whether mutation Y is expected to be contained at a site separate from mutation X. In such a case, it is more desirable to respond by the following means.

That is, in one embodiment, the present invention relates to the above-described probe set, wherein when there is a possibility that at least one mutation Y other than the mutation X may be contained in the target sequence, One base is the base of the mutation Y without depending on the type of the base of the mutation Y! ) And a universal base Z that forms a base pair with the mutation Y at the site corresponding to the mutation Y. In the case of a probe set comprising a detect probe and a reference probe, the universal base Z can be used in a site corresponding to the mutation Y in the detect probe and the reference probe. By using the universal base in this way, it is possible to detect mutations under the same conditions for target nucleic acids having different mutation configurations without slightly changing the measurement temperature as described above. In addition, the use of universal bases makes it possible to accurately and reliably detect mutations in bases that should be detected without being affected by mutations existing in bases other than the base portion that is originally intended to be detected. Can be detected. In the part containing the universal base, the thermodynamic stability of the duplex is slightly lower than when the base pair is formed. However, this is more stable than when non-specific binding occurs, resulting in the substantial absence of two base mismatches and only the difference between a perfect match and one base mismatch. Only needs to be detected.

Here, the universal base is a base that can replace a normal base (adenine, guanine, cytosine, thymine, and peracil) in a nucleic acid sequence, is a base that is complementary to any base, and is adjacent to both bases (or If the universal base is at the end of the sequence, the second base from the end does not significantly destabilize the base pair that forms with the corresponding nucleic acid, and Is a base that does not destroy, and retains the biochemical function of the entire sequence containing it. Therefore, the universal base is almost equally stable even when adenine, guanine, cytosine, thymine, and peracil are contained at the corresponding position in the target sequence to which the sequence containing the universal base hybridizes. A sequence that forms a chemical bond between them and contains the universal base can serve as a substrate for various modifying enzymes (kinases, ligases, etc.). Specific examples of the universal base include, for example, those selected from the group consisting of inosine, 5-nitroindole, and 3-nitropyrrole. In addition, as long as the compound other than these compounds satisfies the above requirements, it can be used as a universal base in the same manner.

The use of a universal base in the present invention is also advantageous in terms of the number of probes required for mutation or polymorphism analysis. For example, when trying to detect two adjacent mutations simultaneously, using a conventional probe set, a total of 16 types of probes were required as shown in Figure 3. In this figure, A, T, G, and C are assigned to a position corresponding to mutation X and a position corresponding to mutation Y, respectively, for a target nucleic acid in which mutation X is G and mutation Y is C. A total of 16 types of probes are prepared. Figure 4 shows an example of the results of an experiment performed using such a probe set. When a total of 16 types of probes are prepared for two sites in this manner, the Tm value of each probe will vary, and a probe that perfectly matches both mutation X and mutation Y will be targeted. The signal from the hybrid formed with the nucleic acid is not always the maximum. As a result, errors may occur in mutation analysis.

On the other hand, when universal bases are used in the present invention, it is only necessary to prepare a total of eight types of probe sets as shown in FIG. Figure 6 shows an example of the experimental results when using these eight types of probe sets. The presence of universal bases in the probe makes it possible to minimize the difference in Tm between each probe without being affected by mutations other than the mutation to be detected.> The target nucleic acid can be more clearly distinguished from the one that does not exactly match the target nucleic acid.

In the above description, the case of two mutations has been described. However, even in the case of three or more mutations, the perfect match and the incomplete match can be more effectively achieved by using universal bases appropriately by essentially the same principle. Can be clearly distinguished.

That is, in one embodiment, the present invention is used when the target sequence contains a mutation X and a mutation Y ( _p _{= 1} ^ _q) (Q is an integer of 1 or more corresponding to the number of mutations other than the mutation X). (1) the mutation X or the mutation X A probe for detecting a polymorphism, has a complementary base a and the mutation X at portions corresponding to the mutant X, at least one mutation of said mutant Y _{(p =} Bok _q) a portion corresponding to, forming the at least one mutation Y _{(p = 1} ^ _q) for without depending on the type of base the at least one mutation Y _{(p = 1} ^ _q) base and base pair in A probe for detecting a mutation X having a universal base Z; and (2) a mutation for detecting at least one mutation in the mutation Y (p- ^ q) or a polymorphism due to the at least one mutation or A probe for detecting a polymorphism or a probe set for detecting a mutation or a polymorphism, wherein at a site corresponding to at least one mutation in the mutation Y _{(p = 1} ^ _q) , It has a base, the mutant X, and the mutation Y _{(p = 1} ~ _q) in at least one of Providing professional one subset, characterized in that it consists; pro portion or probe sets having a universal base Z to at least one site for site that corresponds to mutations than different. In the above-described (1) probe for detecting mutation X, a portion corresponding to the mutation X has a complementary base a, and a base corresponding to the other mutation Y ( _{p =} _toq ) site. If at least one of which is a universal base Z _c this is for a universal base Z is inosine, 5-nitroindole, and 3 - can be selected from the group consisting of two Toropiro Ichiru.

In one (2) mutation detection probes or mutation detection probe set, the portion corresponding to at least one mutation in the mutant Y _{(p =} Bok _q), having a complementary base and base of the mutation , the remaining mutation (the mutation X, and the mutation Y _(p ₌ Bok _q) at least one mutation other than mutation in) and have a base Nitsu sites corresponding to sites at least one of which is a universal base Z. In this case, the UNIVA-Sal base Z can be selected from the group consisting of inosine, 5-nitroindole, and 3-nitropyrrol.

As an alternative to a probe set containing a universal base, when there is a possibility that a mutation Y other than mutation X or Y ( _{p = 1} to _q ) is included in the target sequence, probe, the mutation Y, or Υ in at least a portion corresponding to one of _{([rho =} Bok _q), adenine, Guanin, pro one moistened randomly even cytosine, and any nucleotide Chi Minh In the assembly of molecules One of the features is to use it as a probe. In this case, since probe molecules contain adenine, guanine, cytosine, and thymine randomly, that is, in amounts that are not statistically significantly different, any of the probe molecular species is It will hybridize specifically at the site and be detected.

In the probe set of the present invention, between the above-mentioned mutation X and at least one of the above-mentioned mutations Y or Y (p-hq) (where the mutation is detected by a probe) It is preferable that 0 to 10 bases be present. In such a case, mutation X, and mutant Y, or Υ the center of the at no less one mutation (a one to detect mutations by probe) Gapu lobes molecule of _{([rho =} Bok _q) They will be in close proximity. Comparing the case where a plurality of mutations are concentrated at the center of the probe and the case where the mutations are dispersed in each other, that is, when one is at the center and the other is at or near the terminal, In the former case, the difference in hybridization intensity between a perfect match and a single-base mismatch tends to be larger than in the latter case, making it difficult to detect a large number of samples under the same conditions. It seems that it is more difficult to distinguish both by DNA array. However, in the probe set of the present invention, as described above, the difference between a perfect match, a single base mismatch, and a two base mismatch can be distinguished by slightly changing the temperature or using a universal base. It can be done. Therefore, the present invention is considered to exert its effect sufficiently when two or more mutations are close to the center of the probe, which is more difficult with the conventional technology. When the number of bases between adjacent mutations is large, the effect of the universal base is reduced because the presence or absence of the mutation has a small effect on the hybridization intensity, but the present invention is applied when the number of bases is 10 or less. It is particularly effective when the number of bases is 5 or less, and more effective when the number is 3 bases or less.

In the probe set of the present invention, n defining the total number of bases (N = 2n + 1 or N = 2n + 2) in the probe is in the range of 5 to 18, and as a result, The Tm of each probe in the set is within the desired range of +/- 10 ° C. Preferably.

In the DNA array of the present invention, the above-described probe set of the present invention is immobilized. The DNA array used in the examples of the present invention is for a case where a probe is immobilized on a substrate and the type of the probe is specified by its position. However, the DNA array may be used in the mutation or polymorphism detection method of the present invention as long as the DNA array has a configuration capable of detecting a hybridization reaction in a state where a plurality of types of probes can be identified. It is possible. Therefore, in the present invention, the DNA array is not particularly limited as long as the probe is immobilized on a substrate, and examples thereof include glass, silicon wafers, various porous substrates, gels, and microtiter plates. Also includes those in which a probe is immobilized on a substrate such as beads or beads. In the present invention, all of these are called a DNA array. Further, it is preferable to use a probe having a three-dimensional structure for immobilizing the probe, because the detection sensitivity is increased. In the case of a three-dimensional structure, it is easy to raise or lower the temperature of the sample liquid in the DNA chip through which the sample liquid flows, and to change the temperature of the sample liquid to easily detect mutations and polymorphisms. Become. DNA chips using various porous substrates, gels, beads, etc. as substrates can immobilize more probes than 2D substrates, so the concentration of the target substance in the sample solution is lower. Even in this case, detection is possible, so that a small amount of a sample may be used, which is preferable in that the burden on the subject is reduced.

Since the sequence of the target nucleic acid to be subjected to mutation analysis using a DNA array is generally diverse, the Tm value of a probe for detecting and discriminating those sequences at a single base level significantly is significant. Are generally very different. However, according to the present invention, as described above, the value of n that defines the total number of bases of the probe is set within the range of 5 to 18, so that the Tm value of each probe can be adjusted by this adjustment. It becomes possible. In adjusting the Tm value, it is preferable that the Tm value of all the probes in the probe set to be immobilized on the DNA array be within a desired value range of +/- 10 ° C. . If the Tm values of all probes fall within this range, the hybridization conditions for each probe will be essentially the same. It can be said that the matter is optimal.

In the following, an embodiment of the present invention will be described.

The nucleotide sequence shown in SEQ ID NO: 1 in Table 4 shows the wild-type sequence of the target nucleic acid to be detected, and SEQ ID NO: 2 and SEQ ID NO: 3 show the single nucleotide variant including mutation X and mutation Y, respectively. Is shown. Mutation X is a mutation in which the base in the wild type was changed to G, while mutation Y is a mutation in which the base in the wild type was changed to A in T.

Table 4 Target nucleic acid and sequence of α-α

The sequences described in SEQ ID NOS: 15 to 18 in Table 4 are a set of probes for detecting mutation X. The sequence described in SEQ ID NO: 15 is a probe that perfectly matches the mutation X, and SEQ ID NOs: 16 to 18 have a single-base mismatch with the target nucleic acid. Among them, the sequence shown in SEQ ID NO: 16 does not include the mutation X and is a perfect match when it is a wild type. In these probes, a universal site at the position 2 ' Contains base. The universal base can be any of inosine, 5-nitroindole, or 3-nitropyrrole.

The sequences described in SEQ ID NOs: 19 to 22 in Table 4 are a set of probes for detecting the mutation Y. The sequence described in SEQ ID NO: 19 is a probe that perfectly matches the mutation Y. SEQ ID NOs: 20 to 22 are single-base mismatches with the target nucleic acid. Among them, the sequence shown in SEQ ID NO: 20 does not include the mutation Y and perfectly matches when it is a wild type. In these probes, the universal base is contained in the site from the 2nd base 3 ′ of the mutation Y.

Since the probes shown in SEQ ID NOS: 15 to 22 in Table 4 have a common GC content of 40%, the Tm values of these probes are almost the same. The length of the probe is also common.

When these sets of probes are immobilized on a DNA array and the two types of sample nucleic acids (target nucleic acids) shown in Table 5 are subjected to mutation analysis under the same conditions, each probe and the mutation to be detected The relationship between the numbers is as shown in Table 6.

Table 5 Sequence of target nucleic acid in sample

Table 6 With sample: ur / redice 'product mismatch number

As shown in Table 6, the relationship between the target nucleic acid and these probe sets is either a perfect match or a single base mismatch, and there is no two base mismatch. In the case of sample C, it is a mutant containing mutation X but not containing mutation Υ, that is, a wild type. On the other hand, the sample D is a mutant containing both the mutation X and the mutation Υ.

In both cases of samples C and D, the relationship between the target nucleic acid and the probe is limited to the case of a perfect match and the case of a single base mismatch. There is no need to distinguish between a base mismatch and a two base mismatch.

In the analysis, if a fluorescent substance such as FITC is added to the target nucleic acid, the signal can be quantitatively measured. If the measurement is performed under the optimal conditions for perfect match, the signal derived from the perfect match probe is significantly stronger than the signal derived from the imperfect match probe, so that mutation analysis can be performed.

In this embodiment, two adjacent mutations can be analyzed under the same conditions (see SEQ ID NOS: 1 to 3 in Table 4).

The method for producing the probe set of the present invention may use a technique known in the art. In preparation, it is only necessary to introduce an appropriate base (Α; Τ; G; C; universal base; or a random assembly of A, T, G, C) into a site corresponding to the mutation in the target nucleic acid. Any technique known in the technical field may be used for immobilization to the DNA array.

In the method for detecting a mutation or polymorphism of a target nucleic acid according to the present invention, when a combination of a detect probe and a reference probe is not used, the probe set is immobilized on a solid phase. The target nucleic acid is brought into contact with the DNA array under the optimal conditions for hybridization between each probe in the probe set and the target nucleic acid, and the signal from each probe is quantified. Thereafter, the temperature can be arbitrarily increased or decreased by +/− 10 ° C. from the Tm average value of the probes in the probe set, and the signal is further quantified. Here, the optimal conditions for hybridization can be determined based on the Tm value of each probe in the probe set, and the value of n, which defines the total number of bases in the probe, is within the range of 5 to 18. Therefore, the Tm value of each probe is concentrated in a very narrow range, does not include the mutation X that was originally expected, and the DNA array has a single-base mismatch and a two-base mismatch. If the need arises, the signal can be re-measured by raising or lowering the temperature within a very narrow range of +/- 10 ° C from the T m value.

In the method for detecting a mutation or polymorphism in a target nucleic acid according to the present invention, when a combination of a detect probe and a reference probe is used, the target nucleic acid is applied to a DNA array having a probe set immobilized thereon. The hybridization can be carried out by contacting the hybridization between each probe in a set and the target nucleic acid under optimal conditions, and quantifying and comparing signals from the detect probe and the reference probe, respectively. Example

Probe set design

The presence or absence of a single nucleotide mutation (AT £ → AT) in the third base pair of the exon 2 of the repressor gene P53 3 was determined by the two types of cultured cells, namely the human lymphoblastoid cell lines WTK1 and TK6. An attempt was made to detect the type. WTK1 has a single-base mutation (AT → ATA) at the third base pair of exon 237 codon p53 of tumor suppressor gene p53, whereas TK6 is normal (AT: wild-type). First, the base sequence is described in Table 7, starting from the third base pair of p53 7th exon 2337 codon to be detected and containing 10 bases on the 5'-side and 10'-base 3'-side, respectively. Such a mutation detection probe set was designed and immobilized on a DNA chip. Table 7 Keys used in the examples

Preparation of target nucleic acid

Preparation of the target nucleic acid was performed as follows. Ie human lymphoblastoid cell line

Genomic DNAs were prepared from two cell lines, WTK1 and TK6, respectively. A genomic DNA extraction kit from Qiagen was used for the preparation. Next, each of the prepared genomic DNAs was designated as type III, and a DNA fragment consisting of 98 bases in full length including a mutation site as shown in SEQ ID NO: 28 in the center was amplified by PCR using a terminal fluorescent labeling primer. Use about 250 ng of each type DNA DNA, add a 5'-end FITC-labeled primer to 0.8 M, and perform PCR at 94 ° C / 30 seconds → 65 ° C / 30 seconds-72 ° C After performing 40 cycles at / 30 seconds, the reaction product was subjected to removal of the fluorescent-labeled primer from the reaction product using a Qiagen column (QIAquick PCR purification kit) to obtain the target nucleic acid.

Following hybridization and hybridization products in each probe, the terminal fluorescence-labeled PCR product consisting of 98 bases including the mutation site, prepared for WTK1 and TK6 cell lines, was used at a salt concentration of 2XSSPE and a temperature of 55 ^nC. The hybridization reaction was performed under the following conditions. The resulting hybridization image is shown in FIG. In the hybridization images, probe spots indicated as G, C, A, and T have solid-phased reference probe 1, reference probe 3, detect probe, and reference probe 2, respectively. As a result of image analysis, target nucleic acids from wild-type 株 6 strains were isolated. In this case, the fluorescence signal brightness of the reference probe 1 was the highest, while when the target nucleic acid derived from the WTK1 strain having a single nucleotide mutation (AT → ATA) was hybridized, the Because the fluorescence signal brightness was highest, TK6! 5) It was possible to clearly detect the wild type at the 7th exon 23 7 bases, and that the WTK1 had an AT → ATA mutation. Industrial applicability

The present invention provides a probe set that enables accurate detection of mutations in various nucleic acid sequences to be analyzed under the same conditions, and a mutation detection method using the same. The present invention particularly provides a probe set that can clearly identify a plurality of mutations even when they are present in close proximity in a target nucleic acid.

Sequence listing

110> Olympus Optical Co., Ltd.

<120> Probes for Detecting Mutation and SNPs, DNA Arrays with the Probes Immobilized, and Methods of Detecting Mutation and SNPs Using the Same

<130> 03P01006

<160> 28

<210> SEQ ID NO: l

C 211> 32

<212> DNA

<213> artificial sequence <400> 1

atgccagata acttgacgat agccaagaac gc 32

<210> SEQ ID NO: 2

<211> 32

<212> DNA

K 213> artificial sequence K 400> 2

atgccagata actggacgat agccaagaac gc 32

<210> SEQ ID NO: 3

C 211> 32

<212> DNA

<213> artificial sequence C 400> 3

atgccagata acttgtcgat agccaagaac gc 32

<210> SEQ ID N0: 4

C 211> 15

<212> DNA

213> artificial sequence <400> 4

tatcgtccag ttatc 15 ku 210> SEQ ID N0: 5

C 211> 15

<212> DNA

<213> artificial sequence <400> 5

tatcgtcaag ttatc 15 ku 210> SEQ ID N0: 6

C 211> 15

<212> DNA

213> artificial sequence <400> 6

tatcgtctag ttatc 15 <210> SEQ ID N0: 7 <211> 15 ku 212> DNA

<213> artificial sequence <400> 7

tatcgtcgag ttatc 15

<210> SEQ ID N0: 8 <211> 15

<212> DNA

<213> artificial sequence ku 400> 8

gctatcgaca agtta 15

<210> SEQ ID N0: 9 c 211> 15

K 212> DNA

<213> artificial sequence ku 400> 9

gctatcgtca agtta 15 ku 210> SEQ ID NO: 10 ku 211> 15

<212> DNA

<213> artificial sequence <400> 10 gctatcggca agtta 15

<210> SEQ ID N0: 11

<211> 15

<212> DNA

<213> artificial sequence <400> 11

gctatcgcca agtta 15

<210> SEQ ID N0: 12

C 211> 32

K 212> DNA

<213> artificial sequence <400> 12

atgccagata actggacgat agccaagaac gc 32 ku 210> SEQ ID N0: 13

C 211> 32

K 212> DNA

<213> artificial sequence <400> 13

atgccagata actggtcgat agccaagaac gc 32

<210> SEQ ID N0: 14

<211> 15

<212> DNA <213> artificial sequence

<220>

<221> modified—base

<222> modified base 6

223〉 universal bases (i (inosine), 5— nitroindole 2 'deoxynucleoside, 3-nitropyrrole 2' deoxynucleoside)

<400> 14

tatcgnccag ttatc 15

<210> SEQ ID NO: 15

<211> 15

K 212> DNA

213> artificial sequence

<220>

<221> modified— base

K 222> modified base 6

223> universal bases (i (inosine), 5— nitroindole 2 'deoxynucleoside, 3-nitropyrrole 2, deoxynucleoside)

<400> 15

tatcgncaag ttatc 15 ku 210> SEQ ID NO: 16

C 211> 15

<212> DNA

213> artificial sequence

<220>

<221> modified— base ycleoxnosi deu

(yE Gy ve.al Dsaeinosine 2 deoxnuco: niO

ronleside1> Ole:

Φ

05

ypymosine 2 xnculeosidmtrorrol- I

gg atcncta ttatc y.eol axnuceoside

((ypy3 22 umval t>a;mosine12exnleo; doicslae nitrolrrol 222if modie bd C 400> 18

gctatcgacn agtta 15

<210> SEQ ID N0: 19

C 211> 15

K 212> DNA

Ku 213> artmcial sequence

<220>

221> modified—base

K 222> modified base 10

<223> universal bases (i (inosine), 5— nitroindole 2 'deoxynucleoside, 3-nitropyrrole 2 deoxynucleoside) 400> 19

gctatcgtcn agtta 15 ku 210> SEQ ID NO: 20

<211> 15

K 212> DNA

213> artificial sequence

<220>

<221> modified one base

<222> modified base 10

223> universal bases (i (inosine), 5-nitroindole 2, deoxynucleoside, 3-nitropyrrole 2 'deoxynucleoside) 400> 20

gctatcggcn agtta 15 <210> SEQ ID NO: 21

C 211> 15

212> DNA

<213> artificial sequence

<220>

221> modified one base

Gang 222> modified base 10

223〉 universal bases (i (inosine), 5-nitroindole 2 'deoxynucleoside, 3-nitropyrrole 2' deoxynucleoside)

<400> 21

gctatcgccn agtta 15

<210> SEQ ID NO: 22

C 211> 32

K 212> DNA

Ku 213> artificial sequence Kuyanagi> 22

atgccagata actggacgat agccaagaac gc 32 ku 210> SEQ ID NO: 23

C 211> 32.

K 212> DNA

213> artificial sequence <400> 23

atgccagata actggtcgat agccaagaac gc 32 <210> SEQ ID NO: 24 K 211> 21

<212> DNA

<213> artificial sequence <400> 24

aactgttaca tatgtagttg t 21

<210> SEQ ID NO: 25 <211> 21

<212> DNA

2'13> artificial sequence <400> 25

aattgttaca catgtagttg t 21

<210> SEQ ID NO: 26 211> 21

<212> DNA

213> artificial sequence <400> 26

aactgttaca aatgtagttg t 21 ku 210> SEQ ID NO: 27 ku 211> 21

212> DNA

213> artificial sequence C 400> 27

aactgttaca gatgtagttg t 21 ku 210> SEQ ID NO: 28

<211> 98

K 212> DNA

<213> homo sapiens ku 400> 28

tggctctgaa tgtaccacca tccactacaa ctacatgtgt aacagttcct gcatgggcgg 60 catgaaccgg aggcccatcc tcaccatcat cacactgg 98

Claims

The scope of the claims

1. a probe set for detecting a mutation or polymorphism in a nucleotide sequence,

At least one probe in the probe set is

When the total number of bases N of the probe is odd (N == 2n + 1, where n is a natural number; the same applies hereinafter), the target is the base at the center of the probe (n + 1th from both ends). A base a complementary to the mutation X present in the target sequence is arranged, or

If the total number of bases N of the probe is even (N = 2n + 2), then the probe will be assigned to any one of the two bases at the center (n + 1th from the 5 'end and 3' end, respectively). A base a complementary to the mutation X present in the target sequence is arranged;

Both the 5 ′ terminal sequence and the 3 ′ terminal sequence of base a complementary to the mutation X include a sequence that is completely complementary to the target sequence.

A probe set characterized by the following.

2. When there is a possibility that the target sequence may contain at least one mutation Y other than the mutation X, each of the above-mentioned probes can be used without depending on the type of the base of the mutation Y. Has a universal base Z that forms a base pair with the mutation Y

The probe set according to claim 1, wherein:

3. Mutation X and mutation Y ( _{p = 1} to _q ) are probe sets to be used when there are mutations other than mutation X in the target sequence (1 or more integers corresponding to the number of mutations other than mutation X). 1) and (2):

(2) At least one mutation or at least one of the mutations Y ( _{p = l} to _q ) A probe for detecting a mutation or polymorphism for detecting a polymorphism due to one mutation, or a probe set for detecting a mutation or polymorphism,

The mutation Y at portions corresponding to at least one of the mutations _{(p = 1} ~ _q) in having a complementary base and base of the mutation, the mutated X, and the mutation Y _{(p =} Bok _q) A probe or probe set having a universal base z in at least one site corresponding to a mutation other than at least one mutation therein;

A probe set, comprising:

4. The probe set according to claim 2, wherein the universal base Z is selected from the group consisting of inosine, 5-nitroindole, and 3-nitropyrrole.

5. When there is a possibility that at least one mutation Y other than the mutation X is included in the target sequence, each of the above-mentioned probes may be used to generate adenine, guanine, cytosine, and thymine at a site corresponding to the mutation Y. 2. The probe set according to claim 1, wherein the probe set is an aggregate of probe molecules in which each nucleotide is randomly included.

6. If the target sequence contains a mutation X and a mutation Y ( _{p = 1} to ^ (q is an integer of 1 or more corresponding to the number of mutations other than the mutation X), the probe ( _{p = l} to _q ) at a site corresponding to at least one mutation, a probe molecule assembly containing any of adenine, guanine, cytosine, and thymine nucleotides at random. 2. The probe set according to claim 1, wherein

7. The mutations X, and between the mutation Y or the mutation Y _{(p =} Bok _q) least one mutation in, and characterized in that 0 to 1 0 bases are present The probe set according to any one of claims 2 to 6.

8. At least one probe in the probe set according to claim 1 (hereinafter, referred to as a detect probe), and a mismatch is generated at a site corresponding to the mutation X, and in the other region, the mismatch occurs with the detect probe. A probe set comprising at least one probe having the same sequence (hereinafter, referred to as a reference probe).

9. A mismatch occurs in at least one probe (detector probe) in the probe set according to any one of claims 2 to 7 and a site corresponding to the mutation X, and A universal base Z at a site corresponding to at least one of the mutation Y or at least one mutation other than at least one of the mutations Y ( _{p = 1} to _q ); in the other region, A probe set consisting of at least one probe (reference probe) having the same sequence as the detectable probe.

10. The probe set according to claim 9, wherein the universal base Z is selected from the group consisting of inosine, 5-nitroindole, and 3-nitropyrrole.

1 1. At least one probe (detection probe) in the probe set according to any one of claims 2 to 7, and the mutation Y or the mutation Y ( _{p = 1} to _q). At a site corresponding to at least one of the mutations other than at least one of the above, consisting of a probe molecule assembly (reference probe) randomly containing any nucleotide of adenine, guanine, cytosine, and thymine. Probe set.

1 2. n, which defines the total number of bases (N = 2n + 1 or N = 2n + 2) of each probe in the probe set, is in the range of 5 to 18; The probe set according to any one of claims 1 to 7, wherein the Tm of each probe falls within a desired value range of +/- 10 ° C.

1 3. n, which defines the total number of bases (N = 2n + 1 or N = 2n + 2) of each probe in the probe set, is in the range of 5 to 18; The probe set according to any one of claims 8 to 11, wherein the Tm of each probe falls within a desired value range of +/- 10 ° C.

1 4. A DNA array on which the probe set according to any one of claims 1 to 13 is immobilized.

1 5. A DNA array on which the probe set according to any one of claims 1 to 7 and 12 is immobilized, wherein a target nucleic acid is separated from each probe in the probe set and the target nucleic acid. Under optimal conditions for hybridization between Contact, quantitate the signal from each probe, and optionally further raise or lower the temperature by +/- 10 ° C from the average Tm of the probes in the probe set, and then further quantify the signal A method for detecting a mutation or polymorphism in a target nucleic acid, comprising:

16. A DNA array on which the probe set according to any one of claims 8 to 11, and 13 is immobilized, wherein a target nucleic acid is selected from each probe in the probe set and the target nucleic acid. Contacting the hybridization under optimal conditions during the reaction, and quantifying and comparing the signals from the detect probe and the reference probe, respectively. Detection method.